JPH057000B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing aromatic carboxylic acid amide from aromatic nitrile by the action of microorganisms. More specifically, it relates to a method for producing aromatic carboxylic acid amide from aromatic nitrile by the action of microorganisms belonging to the genus Corynebacterium or Genus Nocardia and having the ability to hydrate aromatic nitriles. It is something. Aromatic carboxylic acid amides are compounds useful as raw materials for fine chemicals such as pharmaceuticals and agricultural chemicals.
In particular, pyridine-3-carboxylic acid amide (nicotinic acid amide) is a component of the vitamin B complex, and is an important compound used in medicines, food additives, feed additives, and the like. Various methods are known for producing aromatic carboxylic acid amides, such as the old method using aromatic carboxylic acid as a raw material, and the recent method of catalytic hydration of aromatic nitrile using an alkali catalyst or metal catalyst. However, it has problems such as the preparation of the catalyst and the separation and purification of the produced amide due to the production of by-products, which are complicated. In order to solve these problems, the present inventors focused on a method using microorganisms as a method for producing aromatic carboxylic acid amide from aromatic nitrile under mild conditions and capable of increasing high selectivity. An attempt to obtain aromatic carboxylic acid amide from aromatic nitrile using microorganisms was made in JP-A-51-86186, but it was not satisfactory as an industrial production method due to the large amount of bacterial cells used in relation to the substrate. It's not possible. Under these circumstances, the present inventors searched for a highly active strain with better productivity targeting a wide range of microorganisms, and as a result, a new strain of the genus Corynebacterium (Genus
The present invention was achieved by discovering that microorganisms belonging to the genus Corynebacterium or Genus Nocardia efficiently produce aromatic carboxylic acid amides from aromatic nitriles. That is, the present invention provides the ability to hydrolyze aromatic nitriles by the action of microorganisms belonging to the genus Corynebacterium or Genus Nocardia, which have the ability to hydrolyze aromatic nitriles.
This is a method for producing an aromatic carboxylic acid amide using a microorganism, which is characterized by producing an aromatic carboxylic acid amide from the nitrile. The aromatic nitriles used in the present invention include:
For example, benzene-based nitriles such as benzonitrile and pyridine-based nitriles such as 3-dianopyridine (nicotinonitrile) and 4-cyanopyridine may be selected from any source, but industrially each Preferably, they are prepared by ammoxidation of the corresponding aromatic methyl substituents. Furthermore, the microorganism used in the present invention is a bacterium or actinomycete belonging to the genus Corynebacterium or Gemus Nocardia, and has the ability to produce aromatic carboxylic acid amide from aromatic nitrile. Yes, for example, the Japanese Patent Publication No. 1987-
Corynebacterium genus N-771 described in Publication No. 17918
Strain (Corynebacterium SP・N-771) [Feikoken Bacteria No. 4445], Corynebacterium sp. -775 strain (Nocardia SP/N-775)
No.] etc. can be cited as suitable examples. The mycological properties of these strains are described in the above-mentioned Special Publication No. 1983-
It is disclosed in Publication No. 17918. Generally, one type of these strains is used, but a mixture of two or more types of bacteria may be used, and furthermore, a strain other than the above-mentioned strains having a similar effect may be used in combination. Next, general embodiments of the present invention will be described. (1) Reaction method Specifically, the production of aromatic carboxylic acid amide in the present invention is carried out by, for example, culturing the above-mentioned microorganisms in advance in a large scale in an appropriate medium, separating the microbial cells, or separating and extracting these microorganisms or these microorganisms. Immobilized bacterial cells or immobilized enzymes, etc. obtained by immobilizing enzymes, etc. by various methods such as carrier binding method, crosslinking method, entrapping method, etc.
This can be carried out by contacting an aromatic nitrile in water, physiological saline, or other aqueous medium to produce and accumulate aromatic carboxylic acid amide, and then isolating and collecting the product. In addition, the amide can also be produced by culturing microorganisms in the presence of nitrile, or by adding nitrile to the culture solution after culturing microorganisms. (2) Preparation of bacterial cells The bacterial cells used in the present invention are prepared by culturing them in large quantities in advance in an appropriate medium, collecting the bacteria, and washing them. The medium used at this time may be either synthetic or natural, as long as it contains a suitable amount of carbon sources, nitrogen sources, inorganic salts, and other growth-promoting substances. Carbon sources include glucose and maltose; nitrogen sources include ammonium sulfate and ammonium chloride; organic nutrient sources include yeast extract, meat extract, malt extract, and peptone; and inorganic nutrient sources include phosphate, magnesium, potassium, zinc, and iron. , manganese, etc. are commonly used. These media are adjusted to pH 6-9 and then sterilized by heating or the like. Next, inoculate the bacteria, 20-35
C., preferably 25 to 30.degree. C., for 1 to 5 days under aerobic conditions such as aerated agitation culture or shaking culture. After the culture is completed, the bacterial cells are separated by centrifugation or the like, and then washed with water or a buffer solution with a pH of 6 to 9. Immobilization of bacterial cells can be carried out according to conventional methods using polyacrylamide gel entrapment method, etc., if necessary. (3) Reaction conditions As the reaction medium, an aqueous medium such as water containing the raw material aromatic nitrile, physiological saline, or a buffer solution can be used, but water is preferable in consideration of separation of the product after the reaction is completed. . The concentration of aromatic nitriles in the reaction medium is 0.1-20wt%, preferably 0.5-10wt
%, and there is no problem even if it is not completely dissolved in the reaction medium. Further, a surfactant may be used in combination as a solubilizing agent. The concentration of bacterial cells used in the reaction is usually in the range of 0.05 to 10 wt%. reaction system
PH is 6-10, preferably 7-9, and reaction temperature is 0.
-40°C, preferably 5-30°C. It is preferable to adjust the concentrations of substrate and bacterial cells so that the reaction usually completes in 10 minutes to 8 hours. Excessive reaction leads to by-product of aromatic carboxylic acid and must be avoided. (4) Recovery To separate and recover the aromatic carboxylic acid amide from the reaction mixture, for example, after separating (immobilized) bacterial cells from the reaction mixture by filtration or centrifugation, the reaction medium water is heated to a temperature below 50°C. It may be distilled off under reduced pressure at a temperature of 100 ml, followed by absolute drying, but conventional separation methods such as extraction with an organic solvent such as ethyl acetate may also be used. According to the method of the present invention, highly pure aromatic carboxylic acid amide can be obtained because side reactions are almost prevented. However, if desired, conventional purification methods such as activated carbon treatment, activated clay treatment, ion exchange resin treatment, or recrystallization may be applied. Purification using this method yields higher quality products. Hereinafter, typical examples of the method of the present invention will be shown and more specifically explained, but the present invention is not limited to these Examples in any way. Example 1 () Production of bacterial cells 100 ml of a medium containing 10 g of glucose, 5 g of peptone, 3 g of yeast extract, and 3 g of malt extract and adjusted to pH 7.2 was dispensed into a 500 ml Erlenmeyer flask and heated at 120°C. Pressure sterilized for 15 minutes. One platinum loop of Corynebacterium N-774 strain was inoculated into this medium and cultured with shaking at 30°C for 3 days to produce bacterial cells. The bacterial cells were separated from the culture solution by centrifugation, and then added to a phosphate buffer with a pH of 7.2 for 2 hours.
Washed twice. The water content of the washed bacterial cells thus obtained was about 80%. () Reaction 4.0 g of the washed bacterial cells were added to 100 g of an aqueous solution containing 5% 3-cyano-pyridine and adjusted to pH 8.5 with ammonia, and the mixture was stirred and reacted in a 30°C water bath for 1 hour. After the reaction was completed, a portion of the reaction solution was analyzed using high-performance liquid chromatography, and it was found that it contained 5.86% (conversion rate 100%) of nicotinic acid amide and unreacted 3-
Cyanopyridine and nicotinic acid, a side reaction product, were not observed. Bacterial cells were removed from this reaction solution by centrifugation at 0°C, water was then distilled off at below 50°C, and 5.8g of white crystals were obtained. Identified. The melting point of this nicotinic acid amide is 127~
At 130°C, the yield was almost 100%. Example 2 () Production of bacterial cells Bacterial cells were produced in exactly the same manner as in Example 1 () except that Nocardia strain N-775 was used. The water content of the washed bacterial cells was approximately 85%. () Reaction 1 g of the washed bacterial cells was added to 100 g of an aqueous solution of pH 8.5 containing 1% penzonitrile, and the mixture was stirred and reacted at 30° C. for 1 hour. After the reaction was completed, a portion of the reaction solution was analyzed using gas chromatography, and the result was 1.17% (conversion rate 100%).
of benzamide, unreacted benzonitrile and side reaction product benzoic acid were not observed. After removing the bacterial cells from this reaction solution by centrifugation at 0° C., the mixture was extracted with 100 g of hot benzene to obtain 1.02 g of white crystals. It was identified as benzamide based on the IR spectrum etc. The melting point of this benzamide was 126-128°C, and the yield was 86.8%. Examples 3 to 4 1 g of washed bacterial cells prepared in the same manner as Example 1 was added to 1%
was added to an aqueous solution of PH8.5 containing 2-cyanopyridine or 4-cyanopyridine, and reacted at 30°C for 1 hour with stirring. After the reaction was completed, a portion of the reaction solution was analyzed by high performance liquid chromatography, and the results shown in the following table were obtained. 【table】

Claims (1)

[Claims] 1 Corynebacterium (Genus)
Aromatic carboxylic acid produced by a microorganism characterized in that the microorganism belongs to the genus Corynebacterium or Genus Nocardia and has the ability to hydrate an aromatic nitrile to produce the corresponding aromatic carboxylic acid amide from the nitrile. Method for producing acid amide.